We acknowledge financial support from the American Heart Association Scientist Development Grant, 13SDG14670062 (PWA) and the University of Minnesota Doctoral Dissertation Fellowship (ESH). We also acknowledge the microfabrication resources of the Minnesota Nano Center (MNC) and the image processing resources of the University Imaging Centers (UIC), both at the University of Minnesota. Parts of this work were carried out in the Characterization Facility, University of Minnesota, which receives partial support from NSF through the MRS program.

<ol>
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The authors have nothing to disclose.

Hier präsentieren wir ein Protokoll, das auf zuvor entwickelten vMTF Technologie aufbaut, so dass die erweiterte Experiment mal typisch für chronische Gefäßerkrankungen Wege 1,23,24. Um dies zu erreichen, wir Mikro Genipin, die zuvor gezeigt wurde, daß langfristige Funktionalisierung von PDMS Substrate 11 bereitzustellen, unter Verwendung einer Mikrofluid-Abscheidungstechnik Engineered arteriellen Lamellen mit verbesserten vaskulären Gewebelebensfähigkeit zur Verwendung in MTF Kontraktilitä...

Gefäßerkrankungen, wie zerebraler Vasospasmus 1,2, Bluthochdruck 3 und Atherosklerose 4 langsam entwickeln, sind in der Regel in der Natur chronischer und beinhalten dysfunktionalen Kraft Generation von vaskulären glatten Muskelzellen (VSMCs). Unser Ziel ist es, diese langsam fortschreitenden Gefäßstörungen unter Verwendung von in vitro-Methoden mit feinere Kontrolle der Versuchsbedingungen als in in vivo Modellen zu studieren. Wir haben früher entwickelten Gefäßmuskel Dünnschichten (vMTFs) zum Messen funktionellen Kontraktilität in vitro konstruiert kardiovaskulären Geweben 5, jedoch hat diese Methode auf relativ kurzen Zeitstudien wurde. Hier stellen wir ein Substrat Modifikationstechnik, die unsere früheren vMTF Technik zur langfristigen Messungen ausdehnt. Während das Endothel ist auch in der Gesamtkreislauf-Funktion kritisch, entwickelt arteriellen Lamellen eine nützliche Modellsystem für die Beurteilung von Veränderungen in der GefäßKontraktilität während Fortschreiten der Krankheit. Um einen Funktionskreislauf-Erkrankungen Gewebemodell, sowohl die Struktur und Funktion des arteriellen Lamellen Ingenieur, die Grundkontraktionseinheit des Schiffes, muss mit hoher Wiedergabetreue rekapituliert werden. Arterielle Lamellen sind konzentrisch, in Umfangsrichtung ausgerichteten Blätter kontraktiler VSMCs durch Platten aus Elastin 6 getrennt. Mikrokontaktdrucken von extrazellulärer Matrix (ECM) Proteine ​​auf Polydimethylsiloxan (PDMS) Substrate bereits verwendet wurde, um Führungssignale für die Gewebeorganisation bereitzustellen auszurichtenden kardiovaskuläres Gewebe 5,7-10 nachahmen. Allerdings gemusterte Gewebe mit Mikrokontaktdruck kann die Integrität nach 3-4 Tagen in Kultur zu verlieren, was ihre Anwendbarkeit in der chronischen Toxizität. Dieses Protokoll stellt eine Lösung für dieses Problem durch den Austausch vorherigen Mikrokontaktdruckverfahren mit einer neuen Mikrofluid-Abscheidungstechnik. Genchi et al. Modifizierten PDMS Substraten mit Genipin und found verlängerte Lebensfähigkeit Muskelzellen bis zu einem Monat in der Kultur 11. Hier verwenden wir einen ähnlichen Ansatz zur Kultur von strukturierten glatten Gefäßmuskelzellen auf PDMS zu verlängern. Genipin, ein natürliches hydrolytische Derivat der Früchte Gardenie, ist ein wünschenswerter Kandidat für Substratmodifikation aufgrund seiner relativ geringen Toxizität gegenüber vergleichbaren Vernetzungsmittel und ihre zunehmende Verwendung als Biomaterial in den Gebieten der Gewebereparatur 12,13 und ECM-Modifikation 14, 15. In diesem Protokoll wird Fibronectin als Zellsignalstoff verwendet wird, wie in den vorangegangenen Mikrokontaktdruckverfahren; jedoch Genipin auf PDMS Substrate vor Fibronektin Strukturieren aufgebracht. Somit wird, wie Zellen verschlechtern die gemusterte Matrix neu synthetisierten ECM von angeschlossenen VSMCs können zur Genipin PDMS beschichteten Substrat zu binden. Dieses Protokoll verwendet eine Mikrofluid-Abgabevorrichtung zur zweistufigen Genipin und ECM-Ablagerung. Das Design der mikrofluidischen Vorrichtung ahmt MicroCONTACT Druckmuster für hochentwickelte arteriellen Lamellen in früheren Studien 16 verwendet. Daher erwarten wir, dass dieses Protokoll, um arterielle Lamellen imitiert, die erfolgreich rekapitulieren die in vivo-Struktur und die Kontraktionsfähigkeit der arteriellen Lamellen hochausgerichtet zu erhalten. Wir evaluieren auch Gewebekontraktionsfähigkeit zu bestätigen, dass Genipin ist ein geeignetes Substrat Modifikationsverbindung für langfristige In-vitro-Kreislauf-Erkrankungen Modelle.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Coverslip staining rack</td>
			<td>Electron Microscopy Sciences</td>
			<td><a href="http://www.emsdiasum.com/">www.emsdiasum.com/</a></td>
			<td>72239-04</td>
			<td>Alternative coverslip rack may be used</td>
		</tr>
		<tr>
			<td>Microscope cover glass - 25 mm</td>
			<td>Fisher Scientific, Inc.</td>
			<td><a href="http://www.fishersci.com/">www.fishersci.com</a></td>
			<td>12-545-102</td>
			<td>Alternative brand and size may be used; Microscope slides may also be substituted as substrate base</td>
		</tr>
		<tr>
			<td>Poly(N-iso-propylacrylamide) (PIPAAm)</td>
			<td>Polysciences, Inc.</td>
			<td><a href="http://www.polysciences.com/">www.polysciences.com/</a></td>
			<td>#21458</td>
			<td>Sigma-Aldrich makes an alternate compound, but we have not tested it for use with this protocol; Compound gives strong odor, use proper ventilation</td>
		</tr>
		<tr>
			<td>1-butanol</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/">www.sigmaaldrich.com</a></td>
			<td align="right">360465</td>
			<td>Hazard: flammable (store stock solution in flammable cabinet); flash point is 37 &#176;C, avoid heating; alternative product may be used</td>
		</tr>
		<tr>
			<td>Spincoater</td>
			<td>Specialty Coating Systems, Inc.</td>
			<td><a href="http://www.scscoatings.com/">www.scscoatings.com</a></td>
			<td></td>
			<td>SCS G3P8 Model; Alternative brand and/or model may be used</td>
		</tr>
		<tr>
			<td>Polydimethylsiloxane (PDMS)</td>
			<td>Ellsworth Adhesives (Dow Corning)</td>
			<td><a href="http://www.ellsworth.com/">www.ellsworth.com</a></td>
			<td>184 SIL ELAST KIT 0.5KG</td>
			<td>Alternative distributor may be used</td>
		</tr>
		<tr>
			<td>Fluorescent microbeads</td>
			<td>Polysciences, Inc.</td>
			<td><a href="http://www.polysciences.com/">www.polysciences.com/</a></td>
			<td align="right">17151</td>
			<td>Alternative brand and/or larger size may be used</td>
		</tr>
		<tr>
			<td>Silicon wafers</td>
			<td>Wafer World, Inc.</td>
			<td><a href="http://www.waferworld.com/">www.waferworld.com</a></td>
			<td align="right">2398</td>
			<td>Alternative brand and/or size may be used</td>
		</tr>
		<tr>
			<td>Photoresist&#160;</td>
			<td>MicroChem Corp.</td>
			<td><a href="http://www.microchem.com/">www.microchem.com</a></td>
			<td></td>
			<td>SU-8 3025 allows 20-25-&#181;m feature height</td>
		</tr>
		<tr>
			<td>Contact mask aligner</td>
			<td>Suss MicroTec</td>
			<td><a href="http://www.suss.com/">www.suss.com</a></td>
			<td></td>
			<td>MA6 contact mask aligner; alternative brand and/or model may be used for wafer exposure</td>
		</tr>
		<tr>
			<td>Developer</td>
			<td>MicroChem Corp.</td>
			<td><a href="http://www.microchem.com/">www.microchem.com</a></td>
			<td></td>
			<td>SU-8 Developer; Hazard: flammable</td>
		</tr>
		<tr>
			<td>Tridecafluro-trichlorosilane</td>
			<td>UCT Specialties, Inc.</td>
			<td><a href="http://www.unitedchem.com/">www.unitedchem.com</a></td>
			<td>T2492</td>
			<td>Silane for non-stick coating of patterned silicon wafers (CAUTION: Tridecafluro-trichlorosilane is a flammable and corrosive liquid. Proper personal protective equipment and local exhaust is necessary for use. )</td>
		</tr>
		<tr>
			<td>Surgical biopsy punch</td>
			<td>Integra LifeSciences Corp.</td>
			<td><a href="http://www.miltex.com/">www.miltex.com</a></td>
			<td>33-31AA-P/25</td>
			<td>Alternative brand and/or size may be used</td>
		</tr>
		<tr>
			<td>Genipin</td>
			<td>Cayman Chemical</td>
			<td><a href="http://www.caymanchem.com/">www.caymanchem.com</a></td>
			<td align="right">10010622</td>
			<td>Sigma-Aldrich (G4796-25MG) makes an alternate compound, but we have not tested it for use with this protocol</td>
		</tr>
		<tr>
			<td>1X phosphate buffered saline</td>
			<td>Mediatech, Inc.</td>
			<td><a href="http://www.cellgro.com/">www.cellgro.com</a></td>
			<td>21-031-CV</td>
			<td>Alternative brand may be used</td>
		</tr>
		<tr>
			<td>Fibronectin</td>
			<td>Corning, Inc.</td>
			<td><a href="http://www.corning.com/">www.corning.com</a></td>
			<td align="right">356008</td>
			<td>Sigma-Aldrich (F1056) makes an alternate compound, but we have not tested it for use with this protocol</td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>Life Technologies, Inc.</td>
			<td><a href="http://www.lifetechnologies.com/">www.lifetechnologies.com</a></td>
			<td>15140-122</td>
			<td>Alternative brand and/or size may be used, as long as concentration is the same</td>
		</tr>
		<tr>
			<td>Umbillical artery smooth muscle cells</td>
			<td>Lonza</td>
			<td><a href="http://www.lonza.com/">www.lonza.com</a></td>
			<td>CC-2579</td>
			<td>Alternative cell types may be used for alternative applications. Media should be modified accordingly</td>
		</tr>
		<tr>
			<td>Tyrode&#39;s solution components</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/">www.sigmaaldrich.com</a></td>
			<td>various</td>
			<td>Alternative brand may be used for mixing solution</td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Zeiss</td>
			<td><a href="http://www.zeiss.com/">www.zeiss.com</a></td>
			<td>4350020000000000</td>
			<td>SteREOLumar V12; Alternative brand/type of stereomicroscope may be used</td>
		</tr>
		<tr>
			<td>Temperature-controlled platform</td>
			<td>Warner Instruments</td>
			<td><a href="http://www.warneronline.com/">www.warneronline.com</a></td>
			<td>641659; 640352; 641922</td>
			<td></td>
		</tr>
		<tr>
			<td>Endothelin-1</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/">www.sigmaaldrich.com</a></td>
			<td>E7764-50UG</td>
			<td>Alternative amount may be purchased, as long as treatment concentration is maintained</td>
		</tr>
		<tr>
			<td>HA-1077</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/">www.sigmaaldrich.com</a></td>
			<td>H139-10MG</td>
			<td>Alternative amount may be purchased, as long as treatment concentration is maintained</td>
		</tr>
	</tbody>
</table>

microfluidic genipin deposition technique for extended culture of micropatterned vascular muscular thin films

The chronic nature of vascular disease progression requires the development of experimental techniques that simulate physiologic and pathologic vascular behaviors on disease-relevant time scales. Previously, microcontact printing has been used to fabricate two-dimensional functional arterial mimics through patterning of extracellular matrix protein as guidance cues for tissue organization. Vascular muscular thin films utilized these mimics to assess functional contractility. However, the microcontact printing fabrication technique used typically incorporates hydrophobic PDMS substrates. As the tissue turns over the underlying extracellular matrix, new proteins must undergo a conformational change or denaturing in order to expose hydrophobic amino acid residues to the hydrophobic PDMS surfaces for attachment, resulting in altered matrix protein bioactivity, delamination, and death of the tissues.
Here, we present a microfluidic deposition technique for patterning of the crosslinker compound genipin. Genipin serves as an intermediary between patterned tissues and PDMS substrates, allowing cells to deposit newly-synthesized extracellular matrix protein onto a more hydrophilic surface and remain attached to the PDMS substrates. We also show that extracellular matrix proteins can be patterned directly onto deposited genipin, allowing dictation of engineered tissue structure. Tissues fabricated with this technique show high fidelity in both structural alignment and contractile function of vascular smooth muscle tissue in a vascular muscular thin film model. This technique can be extended using other cell types and provides the framework for future study of chronic tissue- and organ-level functionality.

Das primäre Ziel dieser Arbeit war es, die Überlebensfähigkeit der mikro VSMCs auf hydrophoben PDMS Substrate erstrecken. Dies wurde durch Einbringen eines Mikrofluidabgabesystem, um gemusterte Genipin und Fibronektin auf PDMS (Abbildung 1) abzuscheiden bewerkstelligt. Ablagerung von ECM Proteinen unter Verwendung von Mikrofluid-Liefer ergab High Fidelity Übertragung des Kanalmuster mit bloßen PDMS zwischen Zeilen Genipin und Fibronektin (1D). Die anhaftenden Zellen (1E) fo...

Watch this Scientific Journal Video about Microfluidic Genipin Deposition Technique for Extended Culture of Micropatterned Vascular Muscular Thin Films at JoVE.com

Microfluidic Genipin Deposition Technique for Extended Culture of Micropatterned Vascular Muscular Thin Films

We present a method for microfluidic deposition of patterned genipin and fibronectin on PDMS substrates, allowing extended viability of vascular smooth muscle cell-dense tissues. This tissue fabrication method is combined with previous vascular muscular thin film technology to measure vascular contractility over disease-relevant time courses.

Mikrofluidischen Genipin Abscheidetechnik für Extended Kultur von mikrostrukturierten Vascular Muscular Thin Films

The chronic nature of vascular disease progression requires the development of experimental techniques that simulate physiologic and pathologic vascular ...

microfluidic-genipin-deposition-technique-for-extended-culture

Research

JoVE Journal

Bioengineering

7.6K Aufrufe.  University of Minnesota. We present a method for microfluidic deposition of patterned genipin and fibronectin on PDMS substrates, allowing extended viability of vascular smooth muscle cell-dense tissues. This tissue fabrication method is combined with previous vascular muscular thin film technology to measure vascular contractility over disease-relevant time courses.